Michael Howard said he is "mortified" after breaching hospital hygiene rules on a visit intended to highlight Tory plans to combat superbug MRSA.
Michael Howard is focusing on his plans to tackle MRSA
He washed before entering a ward but failed to repeat the process between shaking hands with two patients.
Earlier on Tuesday he pledged £10m towards measures to speed up diagnosis.
Labour say they are taking action on MRSA and that the number of cases have fallen. Lib Dems say the NHS must make infection control a top priority.
During a visit to London's National Hospital for Neurology and Neurosurgery Mr Howard was careful to clean his hands before entering the Lady Allerton ward but failed to do it again between meeting patients Sallie Hillman and Sophie Merrington.
The hospital infections control nurse said hands should be cleaned before and after touching a patient.
Mr Howard said later: "I went to a hospital earlier on. I must have washed my hands four times but I think I missed out washing them between one patient and another and I should not have done that.
"I was mortified when it was pointed out."
A spokesman for the Tory leader said: "Michael Howard washed his hands with alcohol gel before he entered the ward and at least a further three times when he was on the ward.
"If he inadvertently failed to do so between these two patients he regrets this very much.
"This issue, for very personal reasons, is one he takes extremely seriously."
The Conservative leader lost his mother-in-law to a hospital-acquired infection.
At the morning news conference Mr Howard had said more people died in the UK from hospital-acquired infections than on Britain's roads.
"Hardworking families are paying an extra £5,000 in tax but they are not getting value for money," he said in an attack on Labour's record.
Mr Howard highlighted his party's 10 point action plan to clean up hospitals including empowering doctors and nurses and "bringing back matron".
He said: "A Conservative Government will trust them to exercise their judgement not second guess them at every turn with targets and initiatives."
And he pledged that if elected the Tories would publish all available MRSA infection-rate data by clinical department so people were aware of the dangers they faced and the choices they could make.
"We are committed to urgent, practical action to clean up our hospitals," he pledged. "So if you want to take a stand on cleaner hospitals and send a message to Mr Blair, vote Conservative on 5 May."
Nurses leaders have called on the next government to provide hundreds of thousands of extra uniforms as part of the fight against hospital superbugs.
The Royal College of Nursing, which also launched a 10 point action plan setting out minimum standards for infection control in hospitals, warned poor NHS laundry facilities meant nurses had too few uniforms.
It said this meant staff often had to wash them at home, which may not kill all the bacteria present.'
Lib Dem health spokesman Paul Burstow said the Tories should apologise for their record on MRSA when they were in government.
"What the Conservatives really need to say is sorry. Sorry that they failed to bring back matron while they were in office. Sorry that MRSA rates rocketed in the nineties, and that they failed to collect proper information on hospital infections," he said.
"Instead of offering a real alternative for the NHS, the Conservatives have spent the campaign running the NHS and its staff down."
Cases of people in England contracting the MRSA superbug reached 3,700 between April and September in 2003, but fell back 6% to 3,500 the following year.
Mr Howard was quizzed by journalists about his apparent admission that his party were "two goals down at half-time" in the election campaign.
"What I said about that was that I went to the Carling Cup final a few weeks ago. I went with my son.
"It was a very enjoyable game for 80 minutes because my team [Liverpool] were ahead but in the last 10 minutes I'm afraid it all turned and we were screwed.
"I was just using that metaphor, I'm sorry if people took it the wrong way."
He added: "I am convinced we can win this election."
Ex-Tory leader William Hague said Mr Howard was feeling confident about the election.
"I know he is in a very upbeat mood because I spoke to him last night and he is extremely optimistic and confident and enjoying the campaign," he told BBC News.
"And he should be, because it is a completely different situation from the last two elections."